CN1841066B - Use of il-18 inhibitors for the treatment and/or prevention of atherosclerosis - Google Patents

Abstract

The invention relates to the use of an IL-18 inhibitor for the manufacture of a medicament for the treatment and/or prevention of atherosclerosis.

Description

The application of IL-18 suppressant in treating and/or preventing atherosclerotic

The application is to be April 30 calendar year 2001 the applying date, and application number is dividing an application of 01809054.0 application for a patent for invention " application of IL-18 suppressant in treating and/or preventing atherosclerotic ".

Technical field

The present invention relates to the angiosis field.More particularly, the present invention relates to the application of IL-8 suppressant in treating and/or preventing atherosclerotic.

Background technology

Atherosclerotic is the most common and topmost angiosis, also has many other angiosises certainly.Atherosclerotic mainly influences greatly, medium sized artery, and its pathology comprises fatty streak, dissolves fibrous plaque (fibrolyticplaques) and complication pathology.Atherosclerotic is a kind of chronic inflammation of arterial wall, is characterized in lipid, and like cholesterol, cell like macrophage, T lymphocyte or smooth muscle cell, reaches building up of extracellular matrix (1).Gather in a large number and be called atheroma or patch, they contain calcium usually.Adipose tissue erodable arterial wall reduces the elasticity of artery, and influences blood flow.At last, gather at spot and to form clot on every side, further hinder blood flow, so that artery-clogging fully.Atherosclerotic usually with high-level low-density lipoprotein (LDL)-cholesterol, Lp (a) fibrinogen and factor VII and low-level HDL (LDL)-cholesterol-associated.That dangerous factor comprises is older, the male sex, smoking, diabetes, obesity, hypercholesterolemia, high fat diet and a guy and family history heart disease.Organ property ischaemic like miocardial infarction, is a main cause.

Atheroma is the modal pathology of artery, can further concurrent thromboembolism.Atheromatous plaque often makes arterial lumen narrow down, thereby causes ischaemic, also can cause the Telatrophy in blood perfusion district sometimes.Hypertension that serious consequence comprises the heart failure that causes owing to the angina pectoris that myocardial ischemia causes, owing to ischaemic or non-ischemic incident, cause owing to RAS and kidney are replied renin secretion on physiology increase the blood perfusion that occurs.

Atherosclerotic and artery sclerosis refer to separate pathological conditions sometimes; Atherosclerotic of the present invention is defined as concrete because atheroma makes artery sclerosis or follows the string, and artery sclerosis is meant the artery sclerosis that is caused by any one reason or follows the string.

Atherosclerotic complication or consequence comprise coronary artery disease (coronary atherosclerosis), cause blood supply insufficiency (ischaemic/angina pectoris), acute myocardial infarction (miocardial infarction, heart disease are sent out), TIA (TIA) or apoplexy and to the infringement of blood vessel, muscle or organ owing to blocking.

Aneurysm is the permanent unusual expansion of blood vessel, also is the atherosclerotic general origin cause of formation.The atherosclerotic abdominal aneurvsm usually occurs on one's body the old patient.Their entering retroperitoneal spaces of can breaking.In the atherosclerotic aneurysm,, make the snapback fibre fracture because the muscle ischemic in sustainer middle level so tend to lose the great number of elastic tissue and make the media fiberization, discharges the macrophage enzyme subsequently.

Treatment or the atherosis recommendation medicine of prevention of arterial comprise minimizing blood fat/cholesterol.Specifically, extensively adopt reduction LDL-cholesterol cure.At present, that the most frequently used is Si Datingsi (statins), and it is the specific inhibitor of HMG CoA reductase.The other lipid lowering agent comprises such as cholestyramine (cholestyramine), Colestid (colestipol), nicotinic acid, Gemfibrozil (gemfibrozil), probacol, Mevacor medicines such as (lovastatin).

Another kind method is on the atherosclerotic lesion that has generated, to make thrombotic danger reduce to minimum.Aspirin can be used for reducing the danger that clot forms, seemingly a kind of specific inhibitor of thromboxane A2 of mediated platelet aggregation or anti-coagulants.

Through skin " balloon angioplasty " with the top balloon catheter patch is flattened, and increase the blood that flows through obstruction.This technology be used to open the technological similar of heart arter, but this technology can be applicable in other many arteries of health.Coronary artery stenosis can be with the saphenous vein of the part that is sewn to the active proximal arteries and veins or through cutting the internal mammary artery of the wall of the chest, and it is long-range shunts with arterial anastomosis facies anterior cordis.

(for example: suggestion is gathered (endarterectomy) with surgical removal carotid endarterectomize) in certain situation.

Yet, mainly still treat or control dangerous factor, as keeping low fat, low cholesterol and low salt diet; And note health by expert advice; Treatment and control hypertension, diabetes and other disease reduce body weight and smoking cessation, and normally take exercise to improve heart body and to strengthen circulation.

Atherosclerotic different phase all has inflammatory process (1).Cause that by factors such as comprising low shearing stress, modification lipoprotein and proinflammatory cytokine the endothelium activation is considered to the atherosclerotic first step, and by inflammation control (1).Nearest a lot of research all shows, the interaction between blood vessel and the inflammatory cell in atheroma forms, play a crucial role (1).Particularly, the inflammation prepass that suppresses qualification can reduce atherosclerotic generation (1).

Inflammation atherosclerotic plaque break with thrombosis in also play a leading role (2-5), thereby cause acute ischemic syndrome and associated death thereof (6) to occur.In fact, atherosclerotic serious clinical manifestation comprises that infraction appears in the organ of heart, brain and the influence of other atherosclerotic, mainly is owing on the surface of contact of atherosclerotic plaque that breaks, form thrombus obstruction vessel lumen (3,4).Pathological study shows, rapid wear or unstable patch promptly are prone to the patch that breaks or broken, compares with the stabilize plaque that is difficult for breaking at cell and matrix and forms obviously different (7).Inflammatory cell (macrophage and T lymphocyte) contains a large amount of vulnerable plaques, and these patches comprise the thrombus lipid core, is characterized in that thin fibrous cap loses a large amount of extracellular matrixs (7).

Minimizing is synthetic by the collagen of proinflammatory cytokine IFN γ mediation, and increases the degraded metalloproteinases and all can make fibrous cap attenuation and become fragile (7) derived from the activity of the matrix of macrophage.Easy brittle fiber cap breaks and makes high thrombus lipid core receive the effect of blood circulation, and causes obstructive thrombus to form (1,7).So the density of inflammatory cell is considered to its instable good indicator in given atherosclerotic lesion.

Atheromatosis people's clinical prognosis only depends in part on the size (19,20) of pathology.Generally believe now pathology quality (composition of patch) but not the size can better indicate ischemic event appearance.In fact, atherosclerotic serious clinical manifestation (heart and cerebral infarction) mainly is owing on the surface of contact of atherosclerotic plaque that breaks, form thrombus obstruction vessel lumen (19).Pathological study is verified, and rapid wear or unstable patch promptly are prone to the patch that breaks or broken, mainly is present in the inflammatory cell, and appears and lose smooth muscle cell and collagen quantity (20,21) in a large number.In addition, some patches significantly improve the mortality ratio of apoptotic cell like this, thereby form high thrombus lipid core (13,22).

Proinflammatory cytokine has been participated in inflammatory process.Plain 18 (IL-18) of cytokine interleukin are called as interferon-(IFN-γ) inducible factor (8) at first.It is that T lymphocyte auxiliary cell 1 (Th1) is replied the early signal that produces in the development.IL-18 and IL-12, IL-2, antigen, mitogen and some latent factors one work and induce the generation of IFN-γ.IL-18 also can increase the generation of GM-CSF and IL-2, strengthens the multiplication capacity of the T cell that anti-CD3 induces and increases the lethality of the Fas mediation of NK.(ICE caspase-1) produces from its precursor ripe IL-18 by the L-1 'beta ' converting emzyme.The IL-18 acceptor by at least two in part combines co-operating one-tenth be grouped into.In the T cell that stimulates through mouse IL-12, find height and the low-affinity of IL-18 are combined position (9), this can be assumed to a kind of multichain receptor compound.Up to the present, discern two kinds of receptor subunits, all belonged to IL-1 receptor family (10).The signal transduction of IL-18 comprises activation NF-κ B (11).

Recently, isolate the soluble protein that IL-18 is had high-affinity, and cloned people and mouse cDNA and people's gene (12 from human urine; WO 99/09063).This albumen is named as IL-18 and combines albumen (IL-18BP).

IL-18BP is not the extracellular region territory of one of known IL-18 acceptor, but the natural circulating protein of a kind of secreted.It belongs to a new family of secreted protein, and this family also comprises a few vaccination encoding viral protein (12).IL-18BP expresses (12) by forming in spleen.Urine and recombinant IL-18 combine with the IL-18 with high-affinity specifically, and regulate the biological affinity of IL-18.

The IL-18BP gene is positioned on the human chromosome 11q13, and in the genome sequence of 8.3Kb, does not find to encode strides the extron in film district.Four kinds that on the person, find IL-18BP splice variant or isomeride, called after IL-18BP a, and b, c and d, it is terminal that they all share identical N, but C terminal different (12).

In various cDNA storehouse, find because of the mRNA splicing produces 4 kinds of people and 2 kinds of mouse IL-18BP isomeride, its by expression, purifying and to its combine with in and the bioactive ability of IL-18 carried out assessing (23).People IL-BP isomeride a (IL-18BPa) demonstrates maximum affinity to IL-18, and in conjunction with fast, disassociation is slow, and dissociation constant (K (d)) is 399pM.IL-18BPc also enjoys the Ig district of IL-18BPa except that 29 C end amino acids.The K of IL-18BPc (d) little 10 times (2.94nM).Yet when surpassing 2 volumetric molar concentrations, IL-18BPa and IL-18BPc can neutralize and surpass 95% IL-18.The Ig district of IL-18BPb and IL-18BPd isomeride lack of complete, and lack to combine or in the ability of IL-18.Mouse IL-18BPc has identical Ig district with the IL-18BPd isomeride, when surpassing 2 volumetric molar concentrations, also can neutralize to surpass 95% mouse IL-18.And have with the common C tip type of human il-18 BPa master's mouse IL-18BPd also can in and human il-18.Molecular model recognizes to be had big static and hydrophobicity to mix to combine the position in the Ig district of IL-18BP, this soluble its combines (23) with the high-affinity of part.

Summary of the invention

The present invention is based on such result of study: the IL-18 suppressant has clear and definite beneficial effect to the generation of patch in the atherosclerotic mouse model, the development of patch and the stability of patch.Suppress IL-18 and not only can prevent the formation of aorta pectoralis pathology, and induce the atherosclerotic plaque that has generated to change into and stablize the spot phenotype.

Therefore, the present invention relates to make and prevent and/or treat atherosclerotic medicine with the IL-18 suppressant.The invention still further relates to the methods of treatment that treats and/or prevents the atherosclerotic gene therapy.

Description of drawings

Shown in Figure 1 for Human umbilical vein endothelial cells respectively separately with the cultivation of oxidation lipoprotein, or the histogram of the survival rate after cultivating together with oxidation lipoprotein and IL-18 antibody or IL-18BP.

Comparison for the Western Blot that carries out on the protein extract in atherosclerotic artery and controlled artery shown in Figure 2.In Western Blot, use antibody to IL-18BP (hIL-18BP), IL-18 receptor alpha subunit (hIL18R α), IL-18 (IL-18BP) and apoptotic proteins enzyme (Caspase)-1 (Caspase-1p10).

Shown in Figure 3 is the Ago-Gel of ethidium bromide staining, the RT-PCT analysis result of IL-18 and IL-18BP mDNA in its demonstration atherosclerotic plaque cell.

The representational RT-PCT result who compares with people's α-Ji Dongdanbai (regulation and control) expression that symptom and asymptomatic patch are arranged for IL-18 in the atherosclerotic plaque and IL-18BP shown in Figure 4.

Shown in Figure 5 for being used for the distribution plan of the expression vector of electrotransfer in the mouse muscle.

Shown in Figure 6 is the histogram of atherosclerotic intra-arterial lipid dyeing area.Quantitative computer-assisted image analysis of lipid is gathered.Data are represented (empty plastid n=19, IL-18BP plastid n=14) with mean+SD. ^{* * *}Expression P＜0.0001.

Shown in Figure 7 is with the histogram of IL-18BP treatment back with the comparison of the aortic sinus pathology area of controlled (empty plastid).The quantitative computer-assisted image analysis lesion region.Data are represented (empty plastid n=19, IL-18BP plastid n=14) with mean+SD. ^*Expression P＜0.0001.

The IL-18BP of being shown in Figure 8 treats the influence to pathology inflammatory cell amount.Measure controlled down (the macrophage dyeing n=12 of aortic sinus pathology with quantitative computer-assisted image analysis; T lymphocyte dyeing n=15) or with the number percent of the positive area of mouse (macrophage n=13, T lymphocyte n=12) macrophage (black) of IL-18BP treatment and the quantity (grey square) of every square millimeter of infiltrating T lymphocyte.Data are represented with mean+SD. ^{* *}Expression P＜0.0001.

The IL-18BP of being shown in Figure 9 treats the influence to pathology smooth muscle cell and collagen content.Measure controlled (the smooth muscle cell n=6 down of aortic sinus pathology with quantitative computer-assisted image analysis; Collagen n=11) gathers the number percent of (grey square) with positive area (black) of mouse (smooth muscle cell n=6, the collagen n=13) smooth muscle cell of IL-18BP treatment and collagen.Data are represented with mean+SD. ^*Expression P＜0.05; ^*Expression P＜0.01.

Embodiment

The present invention is based on such result of study: acute coronary syndrome patient's circulation IL-18 level can rise and cause that the unstable carotid atherosclerosis spot of apoplexy can increase the generation of IL-18.In addition, proved already that electrotransfer can prevent that fatty streak produces in the aorta pectoralis in the expression plastid DNA body of IL-18BP coding, and the development of the atherosis spot of aortic sinus late arterial in the well-established atherosclerotic mouse model that slows down.The more important thing is, can induce spot to form with the transfection of IL-18 plastid great change (macrophage, T cell, cell death and lipid content reduce, and smooth muscle cell and collagen content increase) takes place, thereby form stable patch phenotype.These results show that for the first time the IL-18 suppressant plays an important role in generation/development that reduces patch and promotion the stable of patch.

Therefore, the present invention relates to make and treat and/or prevent atherosclerotic medicine with the IL-18 suppressant.

Term in the context of the present invention " prevention ", not only refer to prevent fully a certain influence, also refer to before seizure of disease or the early stage any part or prevent, weaken, reduce, go down or eliminate this influence basically of showing effect.

Term in the context of the present invention " treatment ", refer to any effect useful to the progress of disease, be included in the development that weakens, reduces, goes down after the seizure of disease or eliminate pathology.

Term in the context of the present invention " IL-18 suppressant ", referring to can be to weaken, to reduce or to prevent or block partly, in large quantities or fully that IL-18 produces and/or the mode of effect is regulated any molecule of generation and/or the effect of IL-18.

A kind of suppressant of production can be any, processing synthetic to IL-18 or the ripe molecule that negative effect is arranged.The suppressant that the present invention considers can be; For example, the suppressor of interleukin I L-18 gene expression, reduction stop IL-18mRNA to transcribe or cause the correct folding or part of antisense mRNA, the infringement of this mRNA degraded or stop the protein of the ripe or secretion of IL-18 basically, in case the proteinase of the synthetic IL-18 that can degrade etc.The suppressant of producing can be for example, to stop IL-18 mature C aspase-1 suppressant or ICE suppressant.

The IL-18 function inhibitor can be, for example, and the IL-18 antagonist.Antagonist can with enough affinity and specificity combine or isolate IL-18 molecule itself and part or basically neutralization be responsible for IL-18 or the IL-18 combination (for example, its acceptor) that IL-18 combines with its part.The IL-18 signalling channel that is activated in the cell when antagonist also can be suppressed at the IL-18/ receptors bind.

The IL-18 function inhibitor also can be the molecule of solubility IL-18 acceptor or emulation acceptor; Or the material of blocking-up IL-18 acceptor; Or IL-18 antibody; Like monoclonal antibody, or other material or the molecule that stop IL-18 to combine, thereby weaken or stop the triggering of reacting inside and outside the cell that mediates by IL-18 with its target.

Atherosclerotic is also referred to as artery sclerosis.Term in the context of the present invention " atherosclerotic " comprise that all usually describe the disease or the patient's condition that atherosclerotic artery shows; Wherein fatty material accumulates in vascular wall; Cause blood flow narrow and impaired at last, and form thrombus and cause and break and/or corrode.

According to the present invention, atherosclerotic comprises owing to atheroma (atherosclerotic) and other reason (artery sclerosis) make artery sclerosis and follow the string.The term that this paper uses " atherosclerotic " comprise atherosclerotic pathological condition and atherosclerotic complication or sequelae, at preceding text " background technology " in existing being described in detail.

Progression of atherosclerosis comprises the formation of atherosclerotic plaque and is developed to more and more unstable form.Therefore, the present invention also relates to make and weaken or the medicine of the atherosis development of prevention of arterial with the IL-18 suppressant.

On atherosclerotic plaque, forming thrombus, to cause angiemphraxis be the main cause of heart and cerebral infarction, is one of the most serious sequelae of atherosclerotic.Therefore, the present invention also relates to make the thrombotic medicine that treats and/or prevents atherosclerotic plaque with the IL-18 suppressant.

Spot stability influence atherosclerotic plaque develops into the thrombotic harmful or rapid wear spot of easy initiation.Therefore, the invention further relates to make and prevent and/or treat the unsettled medicine of atherosclerotic plaque with the IL-18 suppressant.

Unstable plaque is broken easily, and spot breaks and can cause thrombosis.Therefore, the invention further relates to the medicine of making the atherosis spot erosion of prevention of arterial or breaking with the IL-18 suppressant.

Spot instability and thrombosis can be caused that the activity of blood coagulation agent alive improves before making by Apoptosis, and this possibly be to cause breaking or atherosclerotic plaque formation thrombus that corrodes and the main cause (13,14) that causes embolism.Prove macrophage in oxidation lipoprotein (oxLDL) inducing culture and endothelial cell apoptosis (15) at present.Such shown in following examples, have found that the IL-18 suppressant can significantly reduce the Apoptosis of being induced by oxidation lipoprotein.

According to the present invention; The unexpected discovery compared with the patient who does not return hospital, recurrent events occurring; Among, progression of atherosclerosis inaccessible repeatedly like death, recurrent ischaemic, blood vessel or the patient that often is admitted to hospital because of heart failure, the IL-18 level in the blood significantly rises.Compare with the patient who survives, this IL-18 level rises particularly evident in dead afterwards patient body.Ischaemic patient and the circulation of non-ischemic patients'blood all have high-level IL-18.

Therefore, the present invention also relates to make the medicine that treats and/or prevents the heart failure recurrence with the IL-18 suppressant.Recurrent events can be any incident behind the heart failure,, progression of atherosclerosis inaccessible repeatedly like death, recurrent ischaemic, blood vessel or often be admitted to hospital because of heart failure.

In one embodiment of the present invention, heart failure is an ischaemic, promptly owing to myocardial ischemia causes heart failure.

In a further advantageous embodiment, heart failure is non-ischaemic, as because the tuberculosis of congestive heart failure takes place again then for systemic hypertension, cardiovascular disease or return back to normally earlier.

In one embodiment of the present invention, the IL-18 suppressant is selected from the group of being made up of following: the antibody of ICE suppressant, anti-IL-18 antibody, anti-any one IL-18 receptor subunit, IL-18 receptor signal channel inhibitor, can be with the antagonist of IL-18 competition and blocking-up IL-18 acceptor and IL-18 conjugated protein, it has isomeride, mutain, fusion, functional derivatives, active fragment or the cyclic transformation derivant of identical activity.

Term used herein " mutain " refers to the congener of IL-18BP; Or the congener of viral IL-18BP; One or more amino acid residues of wherein natural IL-18BP or viral IL-18BP are replaced by the different aminoacids residue; Or disappearance, or added one or more amino acid residues in the native sequences of IL-18BP, viral IL-18BP, but the activity of the product that is produced is compared with wild type IL-18BP or viral IL-18BP not have significantly and is changed.These muteins are by known synthetic and/or anchor point induced-mutation technique, or any other suitable known technology preparation.

Any this a kind of mutain preferably has the amino acid sequence that an IL-18BP fully duplicates or viral IL-18BP fully duplicates, thereby has the activity similar basically with IL-18BP.One of activity of IL-18BP is that it can combine IL-18.As long as this mutain has very big combination activity to IL-18.It just can be used for purifying IL-18, as by the affinity chromatography, thereby thinks that it has the activity similar basically with IL-18BP.Therefore; Can confirm whether certain given mutain has the essentially identical activity with IL-18BP by the normal experiment method; Its method comprises a kind of like this mutain is carried out confirming such as simple sandwich competition experiments (simple sandwich competition assay) whether it can combine with the IL-18 of suitable mark, like radioimmunoassay or EUSA (ELISA).

IL-18BP polypeptide mutain that the present invention can use or viral IL-18BP mutain; Or its code nucleic acid; Comprise one group limited basically with replace peptide or the corresponding sequence of polypeptide; Those of ordinary skills can and guide with conventional method according to instruction as herein described and obtain, and need not too much test.

Advantageous variant in the mutain of the present invention is called as " conservative property " and replaces.The conservative amino acid of IL-18BP polypeptide or albumen or viral IL-18BP replaces can comprise one group of interior synonym amino acid, and it has enough similar plysiochemical characteristic, and the replacement between this group atom will keep the biological function (16) of this molecule.Clearly; In above-mentioned sequence, do not change its function and also can carry out amino acid whose insertion and disappearance; If when particularly this insertion or disappearance only relate to few amino acids,, be preferably below 10 as below 30; And can not remove or replace the amino acid that functional configuration is played a crucial role, like cysteine residues.Protein and mutain that this type disappearance and/or insertion are produced belong to the scope of the invention.

Preferred simultaneous administration is those groups that table 1 is listed; Better simultaneous administration is those groups that table 2 is listed; Best simultaneous administration is those groups that table 3 is listed.

The preferred simultaneous administration of table 1

Amino acid synonym group

Ser Ser，Thr，Gly，Asn

Arg Arg，Gln，Lys，Glu，His

Leu Ile，Phe，Tyr，Met，Val，Leu

Pro Gly，Ala，Thr，Pro

Thr Pro，Ser，Ala，Gly，His，Gln，Thr

Ala Gly，Thr，Pro，Ala

Val Met，Tyr，Phe，Ile，Leu，Val

Gly Ala，Thr，Pro，Ser，Gly

Ile Met，Tyr，Phe，Val，Leu，Ile

Phe Trp，Met，Tyr，Ile，Val，Leu，Phe

Tyr Trp，Met，Phe，Ile，Val，Leu，Tyr

Cys Ser，Thr，Cys

His Glu，Lys，Gln，Thr，Arg，His

Gln Glu，Lys，Asn，His，Thr，Arg，Gln

Asn Gln，Asp，Ser，Asn

Lys Glu，Gln，His，Arg，Lys

Asp Glu，Asn，Asp

Glu Asp，Lys，Asn，Gln，His，Arg，Glu

Met Phe，Ile，Val，Leu，Met

Trp Trp

Table 2 is simultaneous administration better

Amino acid synonym group

Ser Ser

Arg His，Lys，Arg

Leu Leu，Ile，Phe，Met

Pro Ala，Pro

Thr Thr

Ala Pro，Ala

Val Val，Met，Ile

Gly Gly

Ile Ile，Met，Phe，Val，Leu

Phe Met，Tyr，Ile，Leu，Phe

Tyr Phe，Tyr

Cys Cys，Ser

His His，Gln，Arg

Gln Glu，Gln，His

Asn Asp，Asn

Lys Lys，Arg

Asp Asp，Asn

Glu Glu，Gln

Met Met，Phe，Ile，Val，Leu

Trp Trp

The simultaneous administration that table 3 is best

Amino acid synonym group

Ser Ser

Arg Arg

Leu Leu，Ile，Met

Pro Pro

Thr Thr

Ala Ala

Val Val

Gly Gly

Ile Ile，Met，Leu

Phe Phe

Tyr Tyr

Cys Cys，Ser

His His

Gln Gln

Asn Asn

Lys Lys

Asp Asp

Glu Glu

Met Met，Ile，Leu

Trp Met

The embodiment that in protein, produces aminoacid replacement can be used to obtain the IL-18BP polypeptide used among the present invention or the mutain of protein, or the mutain of viral IL-18BP, and it comprises any known method step; United States Patent (USP) 4,959,314,4 like people such as Mark; 588; 585 and 4,737,462; People's such as Koths 5,116,943; People's such as Namen 4,965,195; People such as Chong 4,879,111 with people's such as Lee 5,017,691 patents in the method that proposes, and the lysine that proposes in the United States Patent(USP) No. 4,904,584 people such as () Shaw replaces protein.

Term " fusion " refers to that a polypeptide comprises IL-18BP or viral IL-18BP or its mutain and another protein, and the protein as the residence time in body fluid prolongs merges.So IL-18BP or viral IL-18BP can with another protein, polypeptide etc., like immunoglobulin (Ig) or its fragment, merge.

" functional derivatives " that this paper uses comprises IL-18BP derivant or viral IL-18BP and mutain and fusion, and their available means known in the art are from the functional groups preparation of residue or N end or the side chain existence of C end group group.As long as they are still pharmaceutically acceptable, promptly they do not have destruction and IL-18BP or the similar basically protein active of viral IL-18BP activity, and can not make the composition that contains them have toxicity, include in the present invention.These derivants for example can comprise the polyglycol side chain, and it can cover the antigen position and prolong IL-18BP or the stop of viral IL-18BP in body fluid.Other derivant comprises the carboxyl fatty ester, the O-acyl derivative that the N-acyl derivative of the acid amides that carboxyl and ammonia or primary amine or secondary amine reaction produce, the free amine group of amino acid residue and acyl moiety molecule (like alkanoyl or carbocyclic ring aroyl) formation or free hydroxyl group (like the free hydroxyl group of seryl or threonyl residue) and acyl moiety molecule form.

" active part " of IL-18BP of the present invention or viral IL-18BP, mutain and fusion comprises the fragment or the precursor of independent protein molecular polypeptied chain; Or protein molecular and correlation molecule or coupled residue; Like sugar or phosphate radical residue together; Or the aggregation of protein molecular or saccharide residue self, as long as said part has the activity similar basically with IL-18BP.

In another preferred embodiment of the present invention, the IL-18 suppressant is an IL-18 antibody.Anti-IL-18 antibody can be polyclone or monoclonal, chimeric, peopleization or even the antibody of peopleization fully.The characteristic of recombinant antibodies and fragment thereof is can combine with the IL-18 high-affinity in vivo, and toxicity is low.Can be used for the characteristic that antibody of the present invention should have is patient to be carried out the treatment of enough time, they can be well to going down or mitigate the disease or any symptom or one group symptom relevant excellently with the state of an illness, and toxicity is low.

Through with the IL-18 immunity, be prone in animals such as rabbit, goat or mouse, produce neutralizing antibody.Immune mouse is specially adapted to provide B cell source with the preparation hybridoma, and then the cultivation hybridoma produces a large amount of anti-IL-18 monoclonal antibodies.

Chimeric antibody is to have from two or more sections of different animals species or the immunoglobulin molecules of part.Usually the variable region of chimeric antibody is from non-human mammal antibody, and like mouse monoclonal antibody, and the stable region of this immunoglobulin (Ig) is from the human immunoglobulin(HIg) molecule.These two districts and make up best as conventional method mensuration reduced immunogenicity (24) is arranged.The humanized antibodies is the immunoglobulin molecules that makes up with technique for gene engineering, the comparison displacement mouse stable region of wherein choosing, and keep the former land of mouse-anti.The mouse that is produced-people's chimeric antibody has reduced immunogenicity better, and has improved the pharmacokinetics (25) in human body.

Therefore in another preferred embodiment, IL-18 antibody is peopleization IL-18 antibody, and for example, European patent application EP 0974600 has been narrated the preferred example of the anti-IL-18 antibody of peopleization.

In another preferred embodiment, IL-18 antibody is human antibody.At WO 00/76310, WO 99/53049, US 6,162,963 or AU 5336100 in be described in detail the technology that produces people's antibody.The recombinant antibodies that human antibody preferably produces in the transgenic animals, xenogenesis mouse for example contains the people Ig locus of all or part function.

In a more preferred embodiment of the present invention, the IL-18 suppressant is IL-18BP, or its isomeride, mutain, fusion, functional derivatives, active part or cyclic transformation derivant.These isomeride, mutain, fusion or functional derivatives have kept the biologically active of IL-18BP, particularly combine the activity of IL-18, preferably have the activity of at least one IL-18BP of being substantially similar to.Desirable, the biologically active that these protein have even higher than unmodified IL-18BP.The specific activity IL-18BP of preferred active part is better or have more superiority, and better like stability, toxicity or immunogenicity are lower, or their easier a large amount of productions, or more is prone to purify.

The sequence of IL-18BP and splicing variant/isomeride thereof can obtain from WO 99/09063 or (12) and (23).

Can the functional derivatives of IL-18BP be combined with polymkeric substance to improve the performance of this protein, like stability, half life period, biological utilisation property, human tolerance or immunogenicity.For reaching this purpose, can IL-18BP be connected with polyglycol (PEG).For example, carry out Pegylation by WO 92/13095 said known method.

Therefore, in the preferred embodiment of the present invention, IL-18BP is a Pegylation.

In another preferred embodiment of the present invention, the IL-18 suppressant is a fusion, comprise IL-18 protein-bonded all or part of, itself and all or part of fusion of immunoglobulin (Ig).Those skilled in the art will know that the fusion that is produced has kept the biologically active of IL-18BP, particularly combine the activity of IL-18.This fusion can be directly, or merges mutually through a short delivery couplet peptide, and length is as short as 1-3 amino acid residue or longer, like the length of 13 amino acid residues.Said crosslinking chemical can be E-F-M (Glu-Phe-Met) sequence; Or the tripeptides of 13 amino acid whose crosslinking chemical sequences, it contains the Glu-Phe-Gly-Ala-Gly-Leu-Val-Leu-Gly-Gly-Gln-Phe-Met between IL-18BP sequence and immunoglobulin sequences.The fusion that is produced has improved performance, prolongs like the retention (half life period) at body fluid, and specific activity improves, and expression increases or help the purifying of fusion.

In a preferred embodiment, IL-18BP is blended in the stable region of Ig molecule.Preferably be blended in the heavy chain district, for example, human IgG1's CH2 and CH3 district.The specific fusion proteins that produces comprises the embodiment 11 described IL-18BP of WP99/09063 and the part of immunoglobulin (Ig).Other isomeride of Ig molecule also is fit to be used for producing fusion of the present invention, for example, and isomeride IgG ₂Or IgG ₄, or other Ig class, like IgM or IgA.Fusion can be monomer or polymer, heterogeneous or homogeneity polymer.

Interferon mainly because of its to virus replication and cell proliferation have inhibiting effect by the people cognition.For example, interferon-plays an important role in promoting immunity and inflammatory response.It is said that interferon beta (IFN-β, I type interferon) has anti-inflammatory effect.

The invention still further relates to the application of making the atherosclerotic medicine of treatment that is combined in of IL-18 suppressant and interferon.

Also can interferon be combined with polymkeric substance to improve the stability of this protein, for example, WO 99/55377 has narrated the conjugate between interferon beta and the polyvalent alcohol polyglycol (PEG).

In another preferred embodiment of the present invention, interferon is interferon-beta (IFN-β), is more preferably IFN-β 1a.

IL-18 produce and/or the suppressant of effect preferably with interferon simultaneously, priority or separate use.

In another embodiment of the present invention, the IL-18 suppressant is used in combination with the TNF antagonist.The TNF antagonist is brought into play its activity with several kinds of modes.At first, antagonist can combine or envelope is covered TNF molecule itself with enough affinity and specificity, so that the epi-position of part or neutralized basically TNF epi-position or responsible and TNF receptors bind (below be called " covering property antagonist ").For example, a kind of covering property antagonist antibody that can be anti-TNF.

On the other hand, the TNF antagonist can suppress the TNF signalling channel that TNF combines the back to be activated by cell surface receptor (below be called " signal antagonist ").These two groups of antagonists can be separately or together, is used in combination with the IL-18 suppressant and treats atherosclerotic.

The TNF antagonist is easy to identification and through the conventional method screening and assessment, promptly at external sensitive cell line, can cause like TNF in the human B cell of its propagation and immunoglobulin,exocrine and with candidate antagonist the influence of natural TNF activity estimated.This test comprises different dilutabilitys, for example, test used TNF molar weight 0.1 times-100 times, the TNF preparation of candidate antagonist, and do not have TNF or have only the regulation and control (26) of antagonist.

It is the TNF antagonist that the present invention preferably uses that envelope is covered antagonist.Cover in the antagonist polypeptide that preferably uses those to combine TNF and have reduced immunogenicity with high-affinity in envelope.Especially preferably use the neutralizing antibody of soluble TNF acceptor molecule and anti-TNF.For example, soluble TNF-RI and TNF-RII can be used for the present invention.These truncate shape acceptors are the preferred antagonists of the present invention, and it comprises cell outskirt or its funtion part of acceptor, and for example, EP914431 has narrated truncate soluble TNF-I type and TNF-II receptor.

The TNF acceptor of truncate shape is soluble, and the TNF inhibition that in urine and serum, detects 30kDa and 40kDa combines albumen, and these two kinds of TNF inhibitions combine albumen to be called TBPI and TBPII (27) respectively.According to the present invention, IL-18 suppressant and TNF antagonist and/or interferon can be simultaneously, successively or separate use.

According to the present invention, preferred TNF antagonist is TBPI and TBPII, is used in combination with the IL-18 suppressant.The derivant of this receptor molecule, fragment, section and biologically-active moiety, the acceptor molecule that becomes the present invention to use by functional assembly.This biologically active equivalent or the derivant of acceptor molecule refer to polypeptide portion or the sequence part of this receptor molecule of encoding, and its size is enough and can combine TNF with affinity, so that the interaction of inhibition or sealing and membrane-bound TNF acceptor.

In another preferred embodiment of the present invention, soluble human TNF-RI (TBPI) is the TNF antagonist that the present invention uses.European patent EP 308378, EP398327 and EP433900 have narrated soluble TNF acceptor molecule of natural and reorganization and preparation method thereof.

The IL-18 suppressant can or separate use with tnf inhibitor while, priority.Better be that IL-18 antibody or antiserum are used in combination with the soluble TNF acceptor with TNF inhibition activity.

In another preferred embodiment of the present invention, this medicine also comprises the COX-suppressant, preferred cox 2 inhibitor.The COX suppressant is well known in the art.For example, WO01/00229 has narrated some concrete cox 2 inhibitors.

Thromboxane (thromboxane), particularly thromboxane A2 suppressant are widely used in the treatment atherosclerotic at present.So in another preferred embodiment of the present invention, this medicine also comprises thromboxane inhibitor, thromboxane A2 suppressant particularly can be simultaneously, successively or separately use.According to the present invention, especially preferably be used with aspirin and IL-18 suppressant.

Causing atherosclerotic one of them reason is that lipid concentration is too high in the blood.So in another preferred embodiment of the present invention, this medicine also comprises lipid lowering agent, can simultaneously, successively or separately use.Lipid lowering agent known in the art comprises medicines such as cholestyramine, Colestid, nicotinic acid, Gemfibrozil, probacol.Especially preferably HMG CoA reductase inhibitor is more preferably the reductase inhibitor that is called statins.Statins reductase inhibitor known in the art has a lot, like Simvastain or lovastatin.

In order better to prevent and/or treat atherosclerotic, a preferred embodiment of the present invention relates to low fat and/or low cholesterol and/or low salt diet and is used the IL-18 suppressant.

In one embodiment of the present invention, IL-18 suppressant consumption is approximately the 0.0001-10mg/kg body weight, or about 0.01-5mg/kg body weight, or about 0.1-3mg/kg body weight, or about 1-2mg/kg body weight.In another preferred embodiment, IL-18 suppressant consumption is about 0.1-1000 μ g/kg body weight, or about 1-100 μ g/kg body weight, or about 10-50 μ g/kg body weight.

The invention still further relates to and utilize the expression vector contain IL-18 suppressant coded sequence to prepare to prevent and/or treat atherosclerotic medicine.Promptly adopt gene therapy to treat and/or prevent disease.Advantage is that the IL-18 suppressant is an expressed in situ, receives the tissue of sickness influence or the IL-18 in the cell thereby directly block effectively.

Such as following examples the explanation, show, contain the expression vector of IL-18BP coded sequence at electrotransfer after, the disease mice model can show the effective expression of IL-18BP.

So in a preferred embodiment, expression vector is preferably in intramuscular injection and uses through electrotransfer.

The present invention also considers normally do not expressing in the inadequate cell of amount of IL-18 suppressant or expression inhibitor, and use can be induced and/or the carrier of augment endogenous property generation IL-18 suppressant.This carrier can be included in acting regulating and controlling sequence in the cell of required expression IL-18 suppressant.For example, this type regulating and controlling sequence can be promoter or enhancer.Then, can regulating and controlling sequence linked to each other as operability with gene through in the correct gene seat of homologous recombination with this regulating and controlling sequence quiding gene group, needed expression be promptly induced or is enhanced.This technology is commonly referred to " the endogenous gene activation " (EGA), narration to some extent in WO91/09955.

Those skilled in the art will recognize that also available same technology reduces IL-18 and expresses, promptly import a negative adjusting part,, in the locus of IL-18, thereby cause downward modulation or prevent that IL-18 from expressing like reticent assembly.Those skilled in the art will recognize that this downward modulation or silence that IL-18 expresses are identical with the effect of using the IL-18 suppressant, be used to prevent and/or treat disease.

The invention still further relates to and use, treat and/or prevent atherosclerotic medicine with the cell manufacturing that produces the IL-18 suppressant through genetic modification.

The invention still further relates to and be used in particular for preventing and/or treating atherosclerotic pharmaceutical composition, it comprises the IL-18 suppressant of effective therapeutic dose and the interferon of effective therapeutic dose.As the IL-18 suppressant, said composition can comprise the antibody of caspase-1 suppressant, anti-IL-18 antibody, anti-all IL-18 receptor subunits, the suppressant of IL-18 signalling channel, IL-18 combination albumen, isomeride, mutain, fusion, functional derivatives, active part or its cyclic transformation derivant of competing and blocking the IL-18 antagonist of IL-18 acceptor and have identical activity with IL-18.

The preferred active component of this pharmaceutical composition is above-mentioned IL-18BP and isomeride, mutain, fusion, functional derivatives, active part or cyclic transformation derivant.

The preferred IFN-β of the interferon of this pharmaceutical composition.

In another preferred embodiment, this pharmaceutical composition comprises the IL-18 suppressant of effective therapeutic dose, and can select is an interferon and TNF antagonist.This TNF antagonist can be in the active antibody of TNF, or the truncate TNF receptor fragments of solubility is also referred to as TBPI and TBPII.Pharmaceutical composition of the present invention also can comprise one or more COX suppressant, preferred cox 2 inhibitor.Pharmaceutical composition of the present invention also can comprise thromboxane inhibitor, like aspirin, and/or lipid lowering agent, like statins.

Definition " pharmaceutically acceptable " refer to comprise not can the interferon activity composition the biologically active effect, and to the avirulent any carrier of the host of dispenser.For example,, can these activated proteins be formulated in the carrier with the injection dosage unit form for intestines and stomach dispenser outside, like salt solution, Glucose Liquid, seralbumin and Lin Ge (Ringer) liquid.

Active ingredient in pharmaceutical of the present invention can be used to individuality with the whole bag of tricks.Drug delivery route comprises in the intracutaneous, transdermal (like sustained release preparation), muscle, approach such as in the peritonaeum, in intravenous, subcutaneous, oral, epidural, part and the nose.Also can adopt other effective treatment approach dispenser, for example absorb or impose on patient's (as passing through carrier), cause this active medicine to express in vivo and secrete through will the encode dna molecular of active medicine of gene therapy through epithelium or endothelial tissue.In addition, can other component of protein of the present invention and biologically active drug like pharmaceutically acceptable surfactant, excipient, carrier, dilution and carrier agent etc., be used together.

For outer intestines and stomach (in intravenous, subcutaneous, muscle) dispenser, can reactive protein be mixed with solution, suspending liquid, emulsion or with the outer carrier (like water, salt solution, Glucose Liquid) of pharmaceutically acceptable intestines and stomach and can keep isotonicity (like mannitol) or the adjuvant of chemical stability (like antiseptic and damping fluid) is processed freeze-dried powder.Sterilize to preparation with routine techniques.

The also available conjugation method of reactive protein of the present invention increase molecule in human body half life period and improve its biological utilisation property.For example, as PCT patented claim WO 92/13095 is said, make molecule and polyglycol crosslinked.

Effective therapeutic dose of activated protein is the function of a lot of variablees, comprises antagonist type, the antagonist clinical state (comprising whether keep the active non-toxic level of endogenous IL-18 caters to the need) to the affinity of IL-18, residual toxicity that antagonist shows, route of administration, patient.

" effectively therapeutic dose " is that the IL-18 suppressant suppresses the bioactive amount of IL-18 when using.The dosage that applies single agent or multi-agent to individuality depends on various factors, comprises pharmacokinetics performance, the drug delivery route of IL-18 suppressant, patient's the state of an illness and characteristic (sex, age, body weight, health status and body type size), symptom degree, concurrent treatment, treatment frequency and desired effect.Those skilled in the art have grasped adjustment and have operated fixed dosage range and measure the method that individual IL-18 suppresses in vivo and in vitro.

According to the present invention, the consumption of IL-18 suppressant is about the 0.0001-10mg/kg body weight, or about 0.01-5mg/kg body weight, or about 0.01-5mg/kg body weight, or about 0.1-3mg/kg body weight, or about 1-2mg/kg body weight.Preferred IL-18 suppressant consumption is about 0.1-1000 μ g/kg body weight, or about 1-100 μ/kg body weight or 10-50 μ/kg body weight.

The preferred drug delivery route of the present invention is a subcutaneous route, the more preferably interior dispenser of muscle of the present invention.

In another preferred embodiment, the IL-18 suppressant is every day or every other day uses.

Every day, dose was divided into several doses or with the slowly-releasing administered, with the required result of effective acquisition usually.For the second time or dispenser subsequently can with impose on this individual same dose first or originally, be less than or carry out more than this dosage.Can when seizure of disease or before the outbreak, carry out the second time or dispenser subsequently.

According to the present invention, the IL-18 suppressant can with effective dose treatment prior to, simultaneously or successively with other therapeutic scheme medicine (like multiple pharmaceutical admixtures) is preventative or therapeutic impose on individuality.Active medicine can be used with other therapeutic agent in identical or different composition simultaneously.

The invention still further relates to a kind of atherosclerotic method that treats and/or prevents, it comprises the IL-18 suppressant that applies an effective inhibitory amount to the host that needs are arranged.

The invention still further relates to a kind of atherosclerotic method that treats and/or prevents, it comprises that applying one to the host that needs are arranged comprises IL-18 suppressant coded sequence expression vector.

In a preferred embodiment of the present invention, apply expression vector system, more preferably apply through intramuscular injection.

The invention further relates to and use the diagnostic marker of IL-18 as the serious clinical prognosis of heart failure.Serious clinical prognosis comprises any deterioration of patient condition, as recurring after the miocardial infarction first time, in addition dead.

IL-18 is preferably used as the diagnostic marker of recurrent events behind the heart failure for the first time.The recurrence disease includes, but not limited to that death, recurrent ischaemic, blood vessel are inaccessible repeatedly, progression of atherosclerosis or often be admitted to hospital because of heart failure.

More than narration for the present invention is carried out, with reference to below the embodiment that provides will be more readily understood content of the present invention, but following examples are to be used for setting forth and not mean that limitation of the present invention.

Embodiment

Material and method

Sample

36 patients that carotid endarterectomy is carried out in collection excise 41 human artery's APs getting off.What be used to regulate and control is no atherosclerotic 2 chests and 3 internal mammary arterys (wherein 2 minimum fibrillar muscle thickens), and they take from autopsy or coronary bypass-forming operation.Rapidly these arteries are immersed in the liquid nitrogen, and are stored in-80 ℃.The patch that is used to extract protein and RNA is wanted to clean rapidly earlier before being stored in-80 ℃, and right Hou is immersed in the liquid nitrogen.The patch that is used for immunohistochemistry research placed 4% fresh paraformaldehyde earlier 2 hours; Transfer to again in 30% sucrose-PBS solution; Use the liquid nitrogen IQF then in optimum cutting temperature tissue treatment nutrient culture media, and be stored in-80 ℃ so that the cryogenic thermostat section.Each sample obtains the section of several 8 to 10 μ m, to carry out fabric analysis and immunohistochemistry research.

The patient classification

In order to study potential relation between the unstable sign of IL-18/IL-18BP expression and spot, we are collected in continuous 23 (36 patients are arranged) the individual patients' that carry out endarterectomy between in May, 2000 and August clinical data with random fashion.Doctor system ground report by carrying out endarterectomy checks with the naked eye whether ulcer is arranged in the spot.Like this, we can be divided into ulcer property spot or non-ulcerative spot with spot.In addition, by clinical symptom patient is divided into different two groups.Patient with cerebral ischemia outbreak clinical symptom relevant with carotid artery stenosis has been classified as one group of symptom.These patients carry out endarterectomy in clinical symptom outbreak back 2-66 days (17.6 ± 5.3 days).And those patients that never occur the cerebral ischemia symptom in the arteria carotis district are classified as asymptomatic one group.System's clinical examination of carrying out based on patient to morbidity around coronary artery or its; Detect asymptomatic carotid artery stenosis, have experience operation ultrasonography person to repeat repeatedly to confirm its order of severity with doppler echo cardiotokography (Doppler echography) by one.Although asymptomatic patient has no precedent carotid artery stenosis district ischemic episode; But existing research shows that the cerebral artery Intimectomy is favourable to these patients' the state of an illness, as asymptomatic cerebral atherosclerosis research (ACAS) investigator (Asymptomatic Carotid Atherosclerosis Study investigators) (28) discloses.

Western Blot analyzes

From 12 atherosclerotic plaques and 5 normal arteries of regulation and control, extract protein.Under the liquid nitrogen condition, freezing sample is clayed into power.The ratio of joining 0.3mL solution with every 10mg net weight is suspended in freezing dissolving buffer solution [20mmol/L three hydrochloric acid, 5mmol/L pH 7.5EGTA, 150mmol/L sodium chloride again with powder again; The 20mmol/L glycerophosphate, 10mmol/L sodium fluoride, 1mmol/L sodium orthovanadate; 1% trinitrotoluene X-100; 0.1 %Tween 20,1 μ g/mL Aprotinins, 1mmol/L PMSF; 0.5mmol/L N-methyl sulphonyl-L-phenylalanine chloromethyl ketone (TPCK), 0.5mmol/L N (a)-p-methyl sulphonyl-L-lysine chloromethyl ketone (TLCK)] in.Extract was cultivated on ice 15 minutes, centrifugal then (12000g, 15 minutes, 4 ℃).Keep the supernatant cut that dissolves in detergent, with the protein concentration in the Bio-Rad protein test method balance sample.

In order to analyze IL-18 and IL-18R, extracting solution of protein is boiled 5 minutes, and be contained in 7.5% or 15% SDS-polyacrylamide gel with werstern blot.For IL-18BP; Agreement according to manufacturer; Make from Escherichia coli (E.Coli) (Serono Pharmaceutical Research Institute, Geneva) next recombined human IL-18, rhIL-18 and Affligel 15 (Biorad) coupling of purifying with the 1mg/mL resin.Extracting solution of protein (60 μ g) in 4 ℃ of overnight incubation, is adjusted to the Tween that 500 μ l contain 0.05%PBS with 20 μ l resins on roller.In order to eliminate any non-specific binding; Resin is centrifugal; And with three hydrochloric acid of 10mM pH8,140mM sodium chloride, 0.5% trinitro-toluene-X-100 (Fluka), the washing of 0.5% dehydrocholate; (Boehringer Mannheim) washes once, uses the three salt acid elutions of 50mM pH 8 at last to use Tris, 200mM sodium chloride, 0.05% trinitro-toluene-X-100,0.05%P40 detergent (Fluka), the 2mM CHAPS of 50mM pH 8 again.Then that resin is centrifugal, resin is suspended in the sample buffer again, boiled 5 minutes, be contained at last on 10% the SDS-polyacrylamide NuPAGE gel (Invitrogen).

Through electrophoresis sample is transferred to cellulose nitrate from polyacrylamide gel.Nitrocellulose filter at room temperature was immersed in the TBST solution that contains 5% no fat anhydrous milk [50mmol/L pH 7.5 3 hydrochloric acid, 250mmol/L sodium chloride, and 0.1%Tween salt solution] 2 hours.Then; With these films and goat anti human il-18 and IL-18R (α-chain) polyclonal antibody (1 μ g/ml) (R&D system), mouse anti human IL-18BP monoclonal antibody (monoclonal antibodies 657.27 of 5 μ g/ml) (people such as Corbaz; 2000manuscript submitted), the anti-people caspase-1 of rabbit polyclonal antibody (1 μ g/ml) (A-19, Santa Cruz) is cultivated together.Excessive 200 times of volumetric molar concentration rhIL-18BP-6his (are come from the Chinese hamster ovary cell purifying; Serono PharmaceuticalResearch Institute; Geneva) cultivated 1 hour with 5 μ g/ml monoclonal antibodies 657.27, through the specificity of competition analysis list clonal antibody 657.27 on the band film.According to the working specification that manufacturer provides, cultivate with the HRP that is incorporated into corresponding antibodies again, with chemiluminescent substance positive band is revealed, these bands can with the naked eye be seen with after Hyperfilm ECL (Amersham company) contacts.

Immunohistochemistry

To at room temperature cultivate 30 minutes from the freezing microtome section of 6 atheromatous plaqueses and 1: 10 notmal horse sera or 1: 10 normal goats serum; Wash once with PBS; Cultivate with identification macrophage (DAKO-CD68 with anti-CD 68 main mouse monoclonal antibodies again; KP1), or with anti-human smooth muscle's α-Ji Dongdanbai master mouse monoclonal antibody cultivate with identification smooth muscle cell (1A4, DAKO).In order to discern IL-18 and the IL-18 acceptor in the atheromatous plaques, using dilute concentration is the specificity goat polyclonal antibody (R&D system) of 5 μ g/ml.Monoclonal antibody specific (people such as Corbaz, 2000manuscript submitted) with anti-recombined human IL-18BP isomeride a (H20) detects IL-18BP.After the PBS washing; To cut into slices and following second biotinylated antibody cultivation: dilute concentration is anti-mouse IgG (the Vector Laboratories of 1: 200 biotinylation horse; Inc.); It is used to detect have anti-CD 68, the dyeing of smooth muscle α-Ji Dongdanbai and IL-18BP antibody, and dilute concentration is 1: 200 the anti-goat IgG of biotinylation horse (Vector), it is used to detect anti-IL-18 and anti-IL-18 receptor antibody.Observe immunostaining with affinity element-biotin HRP toning system (Vectastain ABC Kit PK-6100Vector).In order to make negative regulation, use with the irrelevant antibody of homotype coupling but not main antibody makes contiguous slices dyeing.

The RNA preparation

The method according to Chomczynski and Sacchi (29), since the acid guanidinium thiocyanate solution 29 atheroma plaque extract total RNA, and then phenol and chloroform extraction.Purified RNA is dissolved in the water, and measures its concentration through the absorbance log of 260nm.Electrophoresis evaluation RNA integrality on 1% Ago-Gel.According to manufacturer's agreement, synthesize cDNA from the total RNA of 1 μ g with Promega reverse transcription system.

The sxemiquantitative of human il-18 and IL-18BP and real-time (RT)-PCR in human artery's gruel type spot

Contain 1U AmpliTag archaeal dna polymerase (Perkin Elmer Roche, USA), (Amersham USA) with in the 50 μ l cumulative volumes of 50pmole forward and inverse PCR primer carries out sxemiquantitative PCR and reacts 2.5mMdNTP.Reactant by following condition PTC-200Peltiet Effect thermal cycler (MJResearch, cultivate in USA): 94 ℃ of sex change 1 minute, 55 ℃ of annealing 1 minute, 72 ℃ prolong 1 minute.For the amount that guarantees to react with PCR linear phase time PCR product is suitable, at circulation 25,28 and 31 post analysis IL-18BP, IL-18 and beta-actins.Measure the optimum cycle number of times (being respectively 31,28 and 25 takes turns) of the saturated preceding IL-18BP of band, IL-18 and beta-actin.According to reported sequence (AF110799, D49950, X00351) design following PCR primer: IL-18: reverse 5 '-GCGTCACTACACTCAGCTAA-3 ', forward 5 '-GCCTAGAGGTATGGCTGTAA-3 '; IL-18BP: forward 5 '-ACCTGTCTACCTGGAGTGAA-3 ', reverse 5 '-GCACGAAGATAGGAAGTCTG-3 '; Beta-actin: reverse 5 '-GGAGGAGCAATGATCTTGATCTTC-3 '; Forward 5 '-GCTCACCATGGATGATGATATCGC-3 '.In order to get rid of the amplification of possibility contaminated samples genomic DNA, when not having the cDNA template, carry out the PCR reaction.On 1% Ago-Gel, PCR product (10 μ l) is done electrophoretic analysis with 1 * TAE damping fluid.Gel-colored back and 1kb ladder (Gibco) comparatively validate PCR product size.The relative quantification that under UV-lamp, carries out the ethidium bromide staining band with the digital scientific analysis software of Kodak (Kodak) is measured, and the gained result representes the ratio of house-keeping gene (h beta-actin) with target gene (h IL-18BP, h IL-18).

According to reported sequence (AF110799; D49950; NM002046); Use the green RT-PCR primer of SYBR of expressing the following IL-18BP of software design, IL-18 and GAPD (GAPDH) (house-keeping gene regulation and control) from the primer of PE biosystem: IL-18: reverse 5 '-CAGCCGCTTTAGCAGCCA-3 ', forward 5 '-CAAGGAATTGTCTCCCAGTGC-3 '; IL-18BP: reverse 5 '-AACCAGGCTTGAGCGTTCC-3 ', forward 5 '-TCCCATGTCTCTGCTCATTTAGTC-3 '; GAPDH: reverse 5 '-GATGGGATTTCCATTGATGACA-3 ', forward 5 '-CCACCCATGGCAAATTCC-3 '; Introne GAPDH: reverse 5 '-CCTAGTCCCAGGGCTTTGATT-3 ', forward 5 '-CTGTGCTCCCACTCCTGATTTC-3 '.Test specificity and best primer concentration.PCR and the reaction of specificity introne GAPDH primer are polluted to get rid of the genomic DNA that possibly exist.On 3.5% Ago-Gel, the PCR product is done electrophoretic analysis to confirm not having non-specific expansion to grow.Contain AmpErase uracil transglucosylase (Uracil N-Glycosylase with the RT product (the total DNA of 0.5ng) in 5 μ l/ holes, 25 μ l/ holes; Abbreviation UNG) SYBR Green PC R master potpourri (the PE Biosystem of (0.5 unit/hole); CA, USA) and 20 μ l primers (300nM) carry out the green RT-PCR of SYBR.PCR carries out 2 minutes (being used for AmpErase UNG cultivates to remove the uracil of any importing cDNA) at 50 ℃; Carry out 10 minutes (being used to activate Amplitaq Gold) at 90 ℃; On ABI PRISM 7700 detection systems, circulated 15 seconds at 95 ℃ then, 60 ℃ circulated 1 minute.Like this, make reverse transcription cDNA amplification, and measure its Ct (circulation threshold) value.The equal normalization of all Ct values is to house-keeping gene GAPDH.Observe the monospecific DNA band of IL-18, IL-18BP and GAPDH with the gel electrophoresis analysis method.

With " SYBR Green PC R master potpourri " principle that detects in real time combines with double-stranded DNA and cause the fluorescence enhancing based on SYBR is green, thereby can directly detect the PCR product.

Statistical analysis

Data are with mean+SD (mean ± SEM) expression.With the IL-18 level between Mann-Whitney test comparative group, P＜0.05 just thinks that there were significant differences on the statistics.

Embodiment 1: the protection of IL-18 suppressant causes the death of endothelial cell because of oxidation lipoprotein (oxLDL)

Combine albumen or anti-IL-18 antibody to deposit under the situation about being in or be not at IL-18, the Human umbilical vein endothelial cells (HUVEC) of cultivating is contacted 16 hours with oxidation lipoprotein.As shown in Figure 1,83%HUVEC is contacting back death with oxidation lipoprotein.And whole cell survivals are got off with IL-18BP or the cultivation of anti-IL-18 antibody.Cultivate also no discovery with IL-18BP cell death is arranged.And with the survival rate of anti-IL-18 antibody cultured cell be 89%.

This test clearlys show that two kinds of different I L-18 suppressant are to the protection effect owing to the death of the apoptotic cells in the atherosclerotic plaque.

Embodiment 2: the expression of IL-18 protein and endogenous inhibitor IL-18BP thereof in the atherosclerotic plaque

Analyze extracting solution of protein with Western blot from 12 carotid atherosclerosis arteries and 5 normal regulation arteries.The IL-18 protein that comprises activated form is all highly expressed in the atherosis spot of all arteries, and in normal artery (Fig. 2), detects seldom or do not express.Band 1 to 4 is the sample from atherosclerotic plaque, and band 5 to 7 is the sample from normal artery.Interesting is that the testing result of IL-18 activated form is as if relevant with the expression of the caspase-1 activated form of participating in IL-18 processing (Fig. 2 is front-seat).In the atherosis spot of all arteries, also detect IL-18 receptor protein (being the α chain) and significantly express, in contrast to this, normal artery (Fig. 2 second row) has only utmost point low expression level.In addition, though expression is allos (Fig. 2 first row), most of atherosclerotic plaque is all expressed IL-18BP.

Embodiment 3: the celluar localization of IL-18 protein and endogenous inhibitor IL-18BP thereof in the atherosclerotic plaque

In order to confirm the celluar localization of IL-18 protein and IL-18BP, on 6 carotid atherosclerosis spots, carry out immunohistochemistry research.As shown in Figure 2, IL-18 mainly expresses in macrophage, and these cells possibly be the main sources of IL-18 (not shown) in the spot.Also there are a large amount of CD3-positive lymphocytes in these zones.As if yet the T lymphocyte is not participated in the generation of IL-18 directly.Some intimal smooth muscle cells and accidental endothelial cell are also expressed IL-18.Different is although all blood vessels all do not have to express, to detect IL-18BP at the surperficial endothelial cell of spot microvascular and chamber and significantly express.Be rich in the macrophage district at some,, also detect the relatively low IL-18BP expression bigger with inhomogeneity mainly in the extracellular.

Embodiment 4: the expression of IL-18 and IL-18BP mRNA transcript and and the instable relation of spot in the atherosclerotic plaque

Whether express hIL-18 and IL-18BP mRNA in order to measure people's carotid atherosclerosis spot (Fig. 3), on 6 atherosclerotic plaques, carry out sxemiquantitative RT-PCR.Though the amount heterogeneity of IL-18 and IL-18BP mRNA, the atherosis spot of all arteries all detects IL-18 and IL-18BP mRNA.So,, further analyze this 23 atherosclerotic plaques (Fig. 4) with the green RT-PCR method of SYBR for accurately quantitative IL-18 and IL-18BP mRNA expression.The characteristic of these spots is to be divided into symptom is arranged (instability) and asymptomatic (stablizing) patch through clinical and pathologic finding, has been observed visually ulcer or has not had ulcer.Table 4 is briefly listed these patients' Clinical symptoms.Do not distinguish number percent (60%-95%) and the dangerous factor that the arteria carotis diameter reduces between two groups of patients, whether comprise age, diabetes, high cholesterol, hypertension and smoking.

Table 4

¹These patients are the of short duration or lasting cerebral ischemia attack of appearance in 2-66 days before carrying out endarterectomy

²Treat clinical hypertensive patient's number with rescinnamine

³Treat clinical high cholesterol patient's number with lipid lowering agent

Compare with asymptomatic atherosclerotic plaque, find that in Symptomatic atherosclerotic plaque the amount of IL-18 is upwards increased (being respectively 0.67 ± 0.17 and 2.03 ± 0.5) (Fig. 4 A).Statistical analysis shows that the generation of IL-18 obviously increases (p＜0.0074) in Symptomatic spot, and is having symptom to find that the amount of IL-18BP does not have increase, (being respectively 4.64 ± 0.98 and 2.5 ± 0.92) (Fig. 4 B) in to asymptomatic spot.In other words, though have symptom and asymptomatic two groups all to show between IL-18 and the IL-18BP mRNA it is positive function relation, the slope between two groups differs greatly, and (one group of symptom is arranged: slope is 1.16 [0.19-2.14], r ²=0.36, and asymptomatic one group: slope is 4.79 [2.39-7.20], r ²=0.76; P＜0.05).So, relative increase that the IL-18BP of one group of symptom expresses is arranged as if be not enough to compensate the increase that IL-18 expresses.And, owing to ulcer also is considered to cause unsettled one of them reason of spot, so in ulcer or do not have and make further statistical analysis on the ulceration, the result shows that the amount of IL-18 in the ulceration is significantly to adjusted (p＜0.018) (Fig. 4 C).

These data show that the increase that IL-18 expresses in the atherosclerotic plaque is relevant with the spot instability.

Embodiment 5: the development and stabilization property of IL-18BP regulation and control atherosclerotic plaque pathology in disease body inner model

Method

Patient characteristics

, acute ischemic coronary syndrome (UA and miocardial infarction) patient extracts plasma sample in falling ill back 7 days.UA be defined as with Electrocardiographic ischemic change or with coronary artery disease relevant typical chest pain.Miocardial infarction be based on the cardiogram with blood circulation in myocardium enzyme (creatine phosphokinase and troponin) significantly rise relevant ischemic change and the diagnosis made.The non-ischemic patient that same cardiology department goes to a doctor, they do not have the ischemic sign fully.With commercially available kit (MBL, Japan) blood plasma level of mensuration hIL-18.

Mouse IL-18BP expresses the plastid interior electrotransfer of muscle in vivo

Every at a distance from 3 week, express plastid and pcDNA3IL-8BP for 14 14 big 3 IL-18BP of male C57BL/6apoE KO injected in mice of week.Give regulation and control mouse (n=19) injection regulation and control empty plastid.By known method (attendant number is #Q9ZOM9) (23) mouse IL-18BP isomeride d cDNA is separated, and activate cytomegalovirus under the control of son (Invitrogen) its subclone to the EcoR1/Not1 position of mammalian cell expression vector pcDNA3.Shown in Figure 5 for being called the structure of 334.yh.The regulation and control plastid has similar structure, but therapeutic cDNA.(13) as previously mentioned are at the two shin head intramuscular injection IL-18BP or the regulating and expressing plastid (60 μ g) of anesthetized mice.In brief, two stainless steel electrodes of 4.2 to 5.3mm apart are set in the leg both sides, send through skin electric pulse (8 square wave electric pulses of 200V/cm continue 200 milliseconds at 2Hz) through the PS-15 electric impulser.

EUSA (ELISA) detects mouse IL-18BP (mIL-18BP)

Orifice plate uses recombined small-mouse IL-18BP d affinity thing (the 5 μ g/ hole) covering that comes from the rabbit polyclonal antibody purifying to spend the night.Successively (Sigma) detect solubility mIL-18BP with the rabbit polyclonal antibody (0.3 μ g/ml) of the biotinylation recombined small-mouse IL-18BP (Peprotec) of Chinese People's Anti-Japanese Military and Political College's intestinal flora and outer affinity element peroxidase (1/1000).In order to confirm the specificity of mIL-18BP, catch rabbit polyclonal antibody with the test of Western Blot method.The recombined small-mouse IL-18BP d that produces with HEK 293 cells is a standard.The sensitivity of ELISA is 5ng/ml.

Analyze mouse

(13) as previously mentioned obtain cryogenic thermostat section (8 μ m) from aortic sinus, gather so that detect lipid with oil red, detect collagen and carry out immunohistochemical analysis with Sirius red (Sirius Red).(13) as previously mentioned, with specificity master antibody, as anti-mouse macrophage, clone MOMA2 (BioSource), with the anti-smooth muscle cell α-Ji Dongdanbai and the anti-T lymphocyte CD 3 (Dako) of phosphatase alkali conjugation, make these section statinings.Detect the mortality ratio of cell with TUNEL technology (13).With the naked eye count the CD3 positive cell at random.(13) as previously mentioned, with the computing machine assistant images quantitatively (NS15000Microvision) be determined at the positive atherosclerotic plaque of dyeing of aortic sinus and the interior macrophage of aortic area, smooth muscle cell, collagen or TUNEL.Assess the specificity of immunostaining with dyeing with the immunoglobulin (Ig) that matees with non-immune homotype.Assess the specificity of TUNEL with the disappearance of enzyme terminal deoxynucleotidyl transferase.With 10% buffering formalin leap left subclavian artery and arteriorenal aorta pectoralis are solidified, and make lipid gather dyeing with oil red.Then, these sustainers are opened along longitudinal direction, quantitative (NS15000Microvision) calculates the number percent that lipid gathers with the computing machine assistant images.

The result

This research test has proved that hypothesis IL-18/IL-18BP regulation and control are playing a crucial role aspect atherosclerotic and the spot stability two.Measure acute coronary syndrome patient (20 male sex; 18 women; Mean age is 66.2 ± 1.8 years old, and wherein 14 people have UA, and 34 people have miocardial infarction) and non-ischemic regulation and control patient (10 male sex of going to a doctor in same cardiology department; 3 women, the mean age is 60.0 ± 5.2 years old) the IL-18 blood plasma level.Acute coronary syndrome patient's IL-18 blood plasma level is compared remarkable rising with the regulation and control patient and (is respectively 73.0 ± 12.2 and 146.9 ± 17.1pg/ml; P＜0.05); And the cyclical level of IL-18BP just in contrast; Have only slight rising (to be respectively 7.5 ± 2.5 and 20.1 ± 2.7ng/ml, p=0.06).In addition, also find to have the patient of severe ischemic property cardiac dysfunctions and pulmonary edema clinical indication (224.03 ± 39.1pg/ml compares p＜0.001 with regulation and control), its IL-18 level is relevant with the order of severity of disease, and the state of an illness is heavy more, and the IL-18 level is high more.These results of the sick patient's gained of acute coronary and the known high observations of IL-18 level in the atherosclerotic plaque of stroke patient show that all the IL-18/IL-18BP regulation and control possibly play an important role in Atherosclerosis.

For this reason, we are with this hypothesis of apoE stupor (KO) mouse test of spontaneous generation class human artery atherosclerotic lesion.14 14 week big male mice accept the IL-18BP supply through the expression plastid DNA of electrotransfer encoding murine IL-18BPd in the muscle in vivo, and 19 the age suitable regulation and control mouse accept empty plastid.Per 3 weeks repeat the plastid electrotransfer, treated for 9 weeks after, put to death mouse when big in 23 weeks.In having injected the KO mouse of empty plastid, the blood plasma level of mouse IL-18BP is lower than detection limit (5ng/ml).Yet, found that the IL-BP level of blood reached the highest and (after 323.5 ± 100.9ng/ml), two weeks, also measures 127.4 ± 35.4ng/ml in two days behind the injection single IL-18BP plastid.With IL-18BP or empty plastid after 9 week of treatment, the T-CHOL between 2 groups of patients (be respectively 489.4 ± 34.6 and 480.8 ± 36.3mg/dl) with the HDL serum levels (it is little to be respectively 52.3 ± 9.4 and 48.8 ± 5.1mg/dl) difference.Compare for one group with regulation and control, moderately but can significantly increase (being respectively 28.6 ± 0.8g and 31.8 ± 0.9g, p＜0.05) with a treated animal body weight of IL-18BP treatment.

To atherosclerotic influence, promptly descend aorta pectoralis and aortic sinus in two diverse location check IL-18 supply.Select aorta pectoralis to confirm the effect of IL-18BP to fatty streak generation (atheroma formation), this is that the thorax artery atherosclerotic lesion does not almost appear in 14 all big mouse (data not shown goes out) when beginning transfection because of IL-18BP.In 14 all big mouse (data not shown goes out), but find the aortic sinus atherosclerotic lesion, with the development and the complication of these aortic sinus check spots in late period, this is a stable important determinative of spot.The IL-18BP treatment has appreciable impact to the generation and the development of the atherosclerotic lesion of apoE KO mouse.The assay of aorta pectoralis shows that the treatment of IL-18BP lipid is compared with empty plastid treatment, the lipid of mouse gather descend more (Fig. 6).Quantitative computer-assisted image analysis shows that the degree of atherosclerotic lesion lowers 69% (p＜0.0001) (Fig. 6), proves that IL-18 has therapeutic action to atherosclerotic.In addition, and compare,, just can significantly limit development (the area minimizing 24% of spot, p=0.01) (Fig. 7) of aortic sinus atherosclerotic plaque with IL-18BP plastid treatment 9 weeks only with the treatment of empty plastid.

The more important thing is that late period, the composition of pathology was the unsettled main determining factor of spot, through IL-18BP treatment be improved significantly.Atherosclerotic lesion with IL-18BP plastid treatment mouse makes macrophages infiltration significantly reduce by 50% (p＜0.0001) (Fig. 8), and the T lymphocyte reduces by 67% (p＜0.005) (Fig. 8), and smooth muscle cell gathers increase by 2 times (p＜0.05) (Fig. 9).In addition, confirm that these material alterations and collagen content that sick cell is formed significantly increase by 85%, and total lipid content decline is relevant through Sirius red colouring.

So the IL-18 treatment can significantly weaken the inflammatory process of atherosclerotic lesion, and atherosclerotic plaque is tended towards stability.And; In mouse; the inflammation of pathology is formed remarkable minimizing also makes spot inner cell mortality ratio reduce (using the mouse of IL-18BP treatment is 2.9 ± 0.9%, and the regulation and control mouse is 10.5 ± 3.6%, p＜0.05) greatly with IL-18BP treatment; thereby limited the expansion and the thrombosis (Mallat, 1999) of acellular lipid core.

Conclusion

Through making progress the good atherosis mouse model of class human artery, The above results is clear to show that IL-18 and IL-18BP play key effect in generation, the development and stabilization of atherosclerotic plaque certainly.And in order to prevent the formation of aorta pectoralis early lesion, suppressing the IL-18 activity through supply IL-18BP also has appreciable impact to aortic sinus pathology in late period composition, comprises being transformed into and stablizes the spot phenotype.

Atheromatosis people's clinical prognosis only can depend in part on the area of pathology.Existing now extensively common recognition, quality of pathology (composition of spot) rather than area can better be predicted the outbreak of ischemic disease.In fact, the serious clinical manifestation of atherosclerotic (heart and cerebral infarction) mainly is on the surface of contact of atherosclerotic plaque that breaks, to form thrombus artery-clogging (19).Pathological study proved already that rapid wear and unstable plaque were broken easily or broken, mainly existed in the inflammatory cell, and they lose a large amount of smooth muscle cells and collagen quantity (20,21).In addition, these patches significantly improve the mortality ratio of apoptotic cell, thereby form high thrombus lipid core (13,22).It should be noted that in the mouse with the IL-18BP treatment the unstable sign of all these spots all obviously weakens, this shows that the IL-18 signal is the unsettled main determining factor of spot.

We find that acute coronary syndrome patient's circulation IL-18 level raises, and cause the IL-18 that produces in the unstable arteria carotis appearance harden plaque of apoplexy also to increase, and have confirmed the result of apoE KO mouse gained and the relation of human diseases thus more.These discoveries are taken all factors into consideration, confirmed that the activity of IL-18 suppressant can be used as the important new treatment means of prevention and generation of treatment atherosclerotic plaque and restriction spot complication.

Embodiment 6: high-level IL-18 is relevant with heart failure patient's recurrence

With the IL-18 level of IL-18 specific antibody through ELSIA experimental measurement patients serum.

Test 56 ischemic or non-ischemic patients that have or do not have heart failure altogether.

In dead afterwards patient, the IL-18 level is 216.0 ± 41.5pg/ml, and among the patient of existence, the IL-18 level is 112.2 ± 12.2pg/ml (p=0.0018).

In the patient of recurrent events occurring;, progression of atherosclerosis inaccessible repeatedly or often be admitted to hospital because of heart failure like death, recurrent ischaemic, blood vessel; The level of the IL-1 that measures is 165.8 ± 23.8pg/ml; And in the patient of any nothing recurrence, the IL-18 level is 107.7 ± 14.6pg/ml (p=0.03).

These results show that serious clinical prognosis is arranged, as recurrence or even dead, patient's IL-18 level very high.

Measure 16 blood sample IL-18 levels that have or do not have the non-ischemic patient of heart failure.In dead afterwards patient, the IL-18 level is 199.0 ± 34.8pg/ml, and in the patient of still existence, the IL-18 level is 95.3 ± 20.4pg/ml (p=0.09).

It is respectively 146.6 ± 34.4pg/ml and 95.4 ± 23.9pg/ml (p=0.03) with the IL-18 level of not having the patient of recurring that the patient of recurrence is arranged.Though because patient's number is less, the difference of IL-18 level does not reach statistics, but still clear this trend of level rising of seeing IL-18.

In the ischemic patient, dead patient is respectively 214.2 ± 45.9pg/ml and 118.4 ± 12.8pg/ml (p=0.007) with existence patient's IL-18 level.

It is respectively 162.8 ± 24.7pg/ml and 116.2 ± 16.0pg/ml that the IL-18 level that recurs and do not have the patient of recurrence is arranged.

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Sequence table

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gcacgaagat?aggaagtctg 20

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ggaggagcaa?tgatcttgat?cttc 24

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Claims (1)

1.IL-18 be used to make the diagnosis purposes of the antibody of recurrent events behind the heart failure for the first time, said recurrent events is selected from that recurrent ischaemic, blood vessel are inaccessible repeatedly, progression of atherosclerosis and be admitted to hospital once more because of heart failure, said antibody is IL-18 antibody.

Images